Local anesthetics (LAs) block voltage-gated Na+ channels and impede both motor and sensory functions reversibly during local anesthesia. The receptor for LAs in peripheral Na+ channels is therefore an important target for therapeutics. We have shown that drugs effective for chronic pain, such as antidepressants amitriptyline and duloxetine, antiarrhymthic flecainide, antianginal ranolazine, and anticonvulsant mexiletine, all preferentially block persistent late Na+ currents via the LA receptor. Such late Na+ currents that flow through the open state of inactivation-deficient Na+ channels are known to cause chronic pain, as found in inherited Nav1.7 channelopathies. Further evidence indicates that duloxetine and ranolazine at their therapeutic plasma concentrations are more potent in the block of late Nav1.7 Na+ currents than those of skeletal muscle Nav1.4 or cardiac Nav1.5 counterparts. We therefore hypothesize that these therapeutics target the open state of inactivation-deficient Nav1.7 and/or Nav1.8 Na+ channels for their efficacy in pain relief. Our long-term objective is to delimit the unique receptor for these drugs in persistently open Nav1.7 and Nav1.8 Na+ channels. Toward this goal, we plan to apply above pain therapeutics as molecular probes for their receptors in wild-type and inactivation-deficient Nav1.7 and Nav1.8 Na+ channels. Five specific aims will guide this work: 1. measure the resting and inactivated block of Nav1.7 and Nav1.8 Na+ channels by these pain therapeutics, 2, determine if these drugs block persistent late Nav1.7 and/or Nav1.8 Na+ currents selectively, 3, validate the physiological relevance of the potent block of Nav1.7 and Nav1.8 Na+ channels using cultured DRG neurons, 4, identify the unique drug-binding residues in Nav1.7 and Nav1.8 Na+ channels, and 5, use computer modeling to reconstruct this drug binding site within the inner cavity of Nav1.7 and Nav1.8 open Na+ channels. Specifically, we plan to express Nav1.7 and Nav1.8 Na+ channels in mammalian Hek293t cells by transient transfection. The 50% inhibitory drug concentration (IC50) of resting-, open-, and inactivated block will be determined in wild-type and in inactivation-deficient Nav1.7 and Nav1.8 mutant Na+ channels. Validation of drug potency will be conducted using cultured rat DRG neurons with native intracellular ingredients in conjunction with transfection of wild-type or mutant Nav1.7 and Nav1.8 Na+ channels into these cells. Substitutions of drug-binding residues in inactivation-deficient Nav1.7 and Nav1.8 channels will be performed by site-directed mutagenesis and mutants will be subjected to drug screening. Differences in the drug binding site within the inner cavity will be visualized by computer modeling. Together, our studies will reveal how these diverse drugs selectively target the open state of Nav1.7 and/or Nav1.8 Na+ channels via the shared duloxetine/ranolazine/LA receptor at their therapeutic relevant concentrations. Such information will provide new strategies for the development of pain-selective therapeutics. PUBLIC HEALTH RELEVANCE: The local anesthetic receptor in peripheral sodium channels is an important target for clinical drugs used in local anesthesia and in pain management. Recent evidence suggests that pain therapeutics such as duloxetine may act as analgesics to silence persistent late Na+ currents via a duloxetine/local anesthetic shared receptor. Our goals are to delimit such a receptor in peripheral Nav1.7 and Nav1.8 channels, to resolve the drug/receptor interactions during state transitions, and to explore this receptor site for novel pain therapeutics.